The Factor V molecule is an essential co-factor in the blood clotting mechanism. While the activity associated with Factor V was first identified (by virtue of its absence) in a bleeding patient in 1943, Dr. Mann's laboratory was the first to isolate bovine Factor V as a homogeneous protein, (Nesheim et al.. J. Biol. Chem., 254: 508-517 (1979)). Human Factor V was isolated subsequently (Katzmann et al., Proc. Natl. Acad. Sci. USA, 78: 162-166 (1981)). Dr. Mann's laboratory was the first to prepare both polyclonal (Tracy et al., Blood. 60: 59-63 (1982)) and monoclonal antibodies to this protein (Foster et al., Blood, 61: 1060-1067 (1983); Foster et al., J. Biol. Chem., 258: 5608-5613 (1983); and Foster et al., Thromb. Res.28: 649-661 (1982)). In addition, his laboratory has identified the entire amino acid sequence of the molecule (Jenny et al., Proc. Natl. Acad. Sci. USA, 84: 4846-4850 (1987)) in addition to the sites at which it is cleaved by various plasma proteases. (See for example, Jenny et al., supra and Odegaard et al., J. Biol. Chem., 262: 11233-11238 (1987)). Dr. Mann and his coworkers have conducted extensive studies of how Factor V functions and have identified the following features:
A. The protein circulates as a pro-cofactor, which is not functional in coagulation reactions, (Nesheim et al., J. Biol. Chem., 254: 10952-10962 (1979)).
B. The pro-cofactor can be cleaved by thrombin or by Factor Xa (two blood clotting enzymes) to produce the active co-factor, Factor Va, see for example, Nesheim et al., J. Biol. Chem., 254: 1326-1334 (1979); Tracy et al., J. Biol. Chem. 258: 662-669 (1983); Tracy et al., Proc. Natl. Acad. Sci. USA, 80: 2380-2384 (1983) and Foster et al., J. Biol. Chem., 258: 1398--13977(1983).
C. Factor Va can be inactivated by cleavage by the anticoagulant enzyme activated protein C, (Tracy et al., Boood, supra and Canfield et al., Am. Heart Assn., 51st Sci Session, Dallas, Tex., Nov. 13-16, 1978, Circulation 57-58: II, p. 210, Abst. #86, (1978)).
D. The fibrinolytic enzyme plasmin has two effects on Factor V. It can activate Factor V to Factor Va and then inactivate Factor Va. See for example, Omar et al., J. Biol. Chem., 262: 9750-9755 (1987) and Lee et al., Am. Soc. Hemat., 29th Ann. Mtg., Washington, D.C. Dec. 5-8, 1987, as published in Blood, 70: p389a, Abst. #1412 (1987).
It has previously been reported that several discrete Factor V and/or Factor Va peptides are produced by the proteolytic action of various enzymes which are activated during blood clotting or fibrinolytic reactions. These enzymes include thrombin, Factor Xa, activated protein C and plasmin. The peptides produced by the action of these proteases are distinguishable from one another based on their apparent molecular weight, amino acid composition and sequence.
The observation of these Factor V and/or Factor Va peptides in vivo was first accomplished by the infusion of radiolabeled Factor V into dogs and the subsequent identification of the Factor V and/or Va fragments present in plasma by gel electrophoresis and autoradiography. (See for example, Giles et al., J. Clin. Invest. 74: 2219-2225 (1984) and Nesheim et al., supra).
The separation of these Factor V and/or Factor Va peptides has now been accomplished by the present inventors, through the immunochemical detection of the fragments resulting from in vivo proteolysis of endogenous plasma Factor V in patients with disseminated intravascular coagulation (DIC) using Western blotting techniques and a polyclonal anti-human Factor V antibody. See, Rubin et al., Am. Heart Assn. 9th Sci. Session, Dallas, Tex., Nov. 19, 1986, Circulation 74: II, p. 411, Abst. #1639 (1986). Based upon studies conducted in animals and in humans, the present inventors have verified the detectability and correlation between the presence of these Factor V and Factor Va fragments and the extent (or presence) of coronary artery disease states including disseminated intravascular coagulation (DIC) and exercise induced angina pectoris.